Anti-human papillomavirus 16 e7 t cell receptors

ABSTRACT

Disclosed is a synthetic T cell receptor (TCR) having antigenic specificity for an HLA-A2-restricted epitope of human papillomavirus (HPV) 16 E7, E7 11-19 . Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells are also provided. Antibodies, or an antigen binding portion thereof, and pharmaceutical compositions relating to the TCRs of the invention are also provided. Also disclosed are methods of detecting the presence of a condition in a mammal and methods of treating or preventing a condition in a mammal, wherein the condition is cancer, HPV 16 infection, or HPV-positive premalignancy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.17/101,360, filed Nov. 23, 2020, which is a continuation of U.S.application Ser. No. 16/205,631, filed Nov. 30, 2018, now U.S. Pat. No.10,870,687, which is a divisional of U.S. application Ser. No.15/313,673, now U.S. Pat. No. 10,174,098, which is the U.S. nationalstage of PCT/US2015/033129, filed May 29, 2015, which claims the benefitof U.S. Provisional Patent Application No. 62/004,335, filed May 29,2014, each of which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numbersZIABC010984-5 and ZIABC011479 by the National Institutes of Health,National Cancer Institute. The Government has certain rights in thisinvention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 80,888 Byte XML file named “764396.XML,”dated Aug. 1, 2022.

BACKGROUND OF THE INVENTION

The primary cause of some cancer types such as, for example, uterinecervical cancer, is human papillomavirus (HPV) infection. Despiteadvances in treatments such as chemotherapy, the prognosis for manycancers, including HPV-associated cancers, may be poor. Accordingly,there exists an unmet need for additional treatments for cancer,particularly HPV-associated cancers.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides an isolated or purified T cellreceptor (TCR) having antigenic specificity for human papillomavirus(HPV) 16 E7.

Another embodiment of the invention provides a TCR comprising a humanvariable region and a murine constant region, or a functional variant ofthe TCR, wherein the TCR and the functional variant have antigenicspecificity for human papillomavirus (HPV) 16 E7.

The invention further provides related polypeptides and proteins, aswell as related nucleic acids, recombinant expression vectors, hostcells, and populations of cells. Further provided by the invention areantibodies, or antigen binding portions thereof, and pharmaceuticalcompositions relating to the TCRs (including functional portions andfunctional variants thereof) of the invention.

Methods of detecting the presence of a condition in a mammal and methodsof treating or preventing a condition in a mammal, wherein the conditionis cancer, HPV 16 infection, or HPV-positive premalignancy, are furtherprovided by the invention. The inventive method of detecting thepresence of a condition in a mammal comprises (i) contacting a samplecomprising cells of the condition with any of the inventive TCRs(including functional portions and functional variants thereof),polypeptides, proteins, nucleic acids, recombinant expression vectors,host cells, populations of host cells, antibodies, or antigen bindingportions thereof, or pharmaceutical compositions described herein,thereby forming a complex, and (ii) detecting the complex, whereindetection of the complex is indicative of the presence of the conditionin the mammal, wherein the condition is cancer, HPV 16 infection, orHPV-positive premalignancy.

The inventive method of treating or preventing a condition in a mammalcomprises administering to the mammal any of the TCRs (includingfunctional portions and functional variants thereof), polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs(including functional portions and functional variants thereof),polypeptides, proteins described herein, or any host cell or populationof host cells comprising a recombinant vector which encodes any of theTCRs (including functional portions and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a bar graph showing interferon (IFN)-γ (pg/mL) secreted byperipheral blood lymphocytes (PBL) that were transduced with anucleotide sequence encoding a chimeric anti-HPV 16 E7 TCR (shaded bars)upon co-culture with target 293-A2 cells pulsed with HPV 16 E629-38peptide, 293-A2 cells pulsed with HPV 16 E7₁₁₋₁₉ peptide, 624 cellstransduced with a plasmid encoding HPV 16 E6, 624 cells transduced witha plasmid encoding HPV 16 E7, SCC152 cells, SCC90 cells, CaSki cells,Alb cells, Panc1 cells, or SiHa cells. HLA-A2 and HPV-16 E7 expressionby each target cell is indicated in the bottom of FIG. 1 (“+” indicatespositive for expression and “−” indicates negative for expression).Untransduced cells (unshaded bars) were used as a negative control.

FIGS. 2A-2R are dot plots showing the percentage of cells from a firstdonor (A-I) or a second donor (J-R) that were transduced (B-I and K-R)with one of the recombinant expression vectors set forth in Table 1which expressed the following: HLA-A2/E7₁₁₋₁₉ tetramer⁺/m TRBC⁻ (upperleft quadrant), HLA-A2/E7₁₁₋₁₉ tetramer⁺/m TRBC⁺ (upper right quadrant),HLA-A2/E7₁₁₋₁₉ tetramer⁻/m TRBC⁻ (lower left quadrant), andHLA-A2/E7₁₁₋₁₉ tetramer⁻/m TRBC⁺ (lower right quadrant). The numericalpercentages for each quadrant are provided above each dot plot.Untransduced cells (A and J) were used as a negative control.

FIGS. 3A and 3B are graphs showing IFN-γ secretion by effector cellsfrom Donor 1 (A) or Donor 2 (B) that were transduced with one of therecombinant expression vectors set forth in Table 1 upon co-culture withtarget 624, CaSki, SCC90, or SCC152 cells. For each target cell line,the shaded bars (from left to right) correspond to effector cellstransduced with the following vector: chimeric anti-HPV 16 E7 TCR (α/β),Cys-modified TCR (α/β), LVL-modified TCR (α/β), LVL-Cys-modified TCR(α/β), chimeric anti-HPV 16 E7 TCR (β/α), Cys-modified TCR (β/α),LVL-modified TCR (β/α), or LVL-Cys-modified TCR (β/α). Untransducedcells (unshaded bars) were used as a negative control.

FIG. 4 is a graph showing IFN-γ (pg/mL) secreted by PBL that weretransduced with a retroviral vector encoding the LVL-modified TCR (β/α)(SEQ ID NO: 37) (shaded bars) upon co-culture with target 293-A2 cellspulsed with HPV 16 E629-38 peptide, 293-A2 cells pulsed with HPV 16E7₁₁₋₁₉ peptide, 624 cells transduced with a plasmid encoding HPV 16 E6,624 cells transduced with a plasmid encoding HPV 16 E7, SCC152 cells,SCC90 cells, CaSki cells, Ane cells, Alb cells, Panc1 cells, or SiHacells. HLA-A2 and HPV-16 E7 expression by each target cell is indicatedin the bottom of FIG. 4 (“+” indicates positive for expression and “−”indicates negative for expression). Untransduced cells (unshaded bars)were used as a negative control.

FIG. 5 is a graph showing IFN-γ (pg/mL) secreted by PBL that weretransduced with a retroviral vector encoding the LVL-Cys-modified TCR(β/α) (SEQ ID NO: 38) upon co-culture with target 624 cells, SCC90cells, or CaSki cells without antibodies (black bars) or in the presenceof anti-MHC Class I (grey bars) or anti-MHC Class II antibodies(unshaded bars). As controls, PBL were transduced with DMF5 TCR andco-cultured with 624 cells or transduced with anti-MAGE A3 TCR andco-cultured with 526-CIITA cells without antibodies or in the presenceof anti-MHC Class I or anti-MHC Class II antibodies.

FIGS. 6A-6D are graphs showing specific lysis (%) of target cells CaSki(A), SCC90 (B), SCC152 (C), or 624 cells (D) co-cultured with effectorcells transduced with a retroviral vector encoding the LVL-Cys-modifiedTCR (β/α) (SEQ ID NO: 38) (squares) at various effector:target ratios.Untransduced cells (circles) were used as a negative control.

FIGS. 7A and 7B are graphs showing IFN-γ (pg/mL) secreted by PBL thatwere transduced with a retroviral vector encoding the LVL-Cys-modifiedTCR (β/α) (SEQ ID NO: 38) (shaded bars) or the anti-HPV 16 E6 TCR DCA2E6(unshaded bars) upon co-culture with target cells pulsed with variousconcentrations of HPV 16 E7₁₁₋₁₉ peptide (A) or HPV 16 E629-38 peptide(B).

FIG. 8A is a graph showing IFN-γ (pg/mL) secreted by PBL that weretransduced with a retroviral vector encoding the LVL-Cys-modified TCR(β/α) (SEQ ID NO: 38) (black bars) or the DCA2E6 TCR (grey bars) uponco-culture with target 624 cells, Caski cells, SCC90 cells, or SCC152cells. Untransduced cells (unshaded bars) were used as a negativecontrol.

FIG. 8B is a graph showing IFN-γ (pg/mL) secreted by PBL that weretransduced with a retroviral vector encoding the Cys-modified TCR (0/a)(black bars) or the DCA2E6 TCR (grey bars) upon co-culture with target293-A2 cells pulsed with HPV 16 E6₂₉₋₃₈ peptide, 293-A2 cells pulsedwith HPV 16 E7₁₁₋₁₉ peptide, SCC152 cells, SCC90 cells, CaSki cells, Anecells, Alb cells, Panc1 cells, or SiHa cells. Untransduced cells(unshaded bars) were used as a negative control.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides an isolated or purified T cellreceptor (TCR) having antigenic specificity for human papillomavirus(HPV) 16 E7.

HPV 16 is the subtype of HPV that is most commonly associated withmalignancy. Without being bound to a particular theory or mechanism, HPV16 is believed to cause cancer at least partly through the actions ofthe oncoprotein E7, which keeps cancer cells active in the cell divisioncycle through Rb inactivation. HPV 16 E7 is constitutively expressed incancer cells and is not expressed by normal, uninfected human tissues.HPV 16 E7 is expressed in a variety of human cancers including, but notlimited to, cancer of the uterine cervix, oropharynx, anus, anal canal,anorectum, vagina, vulva, and penis.

The inventive TCR (including functional portions and functional variantsthereof) may have antigenic specificity for any HPV 16 E7 protein,polypeptide or peptide. In an embodiment of the invention, the TCR(including functional portions and functional variants thereof) hasantigenic specificity for a HPV 16 E7 protein comprising, consisting of,or consisting essentially of, SEQ ID NO: 1. In a preferred embodiment ofthe invention, the TCR (including functional portions and functionalvariants thereof) has antigenic specificity for a HPV 16 E7₁₁₋₁₉ peptidecomprising, consisting of, or consisting essentially of, YMLDLQPET (SEQID NO: 2).

In an embodiment of the invention, the inventive TCRs (includingfunctional portions and functional variants thereof) are able torecognize HPV 16 E7 in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR (including functional portions and functionalvariants thereof) elicits an immune response upon binding to HPV 16 E7within the context of an MHC class I molecule. The MHC class I moleculecan be any MHC class I molecule known in the art, e.g., HLA-A molecules.In a preferred embodiment of the invention, the MHC class I molecule isan HLA-A2 molecule.

The TCRs (including functional portions and functional variants thereof)of the invention provide many advantages, including when expressed bycells used for adoptive cell transfer. Without being bound by aparticular theory or mechanism, it is believed that because HPV 16 E7 isexpressed by HPV 16-infected cells of multiple cancer types, theinventive TCRs (including functional portions and functional variantsthereof) advantageously provide the ability to destroy cells of multipletypes of HPV 16-associated cancer and, accordingly, treat or preventmultiple types of HPV 16-associated cancer. Additionally, without beingbound to a particular theory or mechanism, it is believed that becausethe HPV 16 E7 protein is expressed only in cancer cells, the inventiveTCRs (including functional portions and functional variants thereof)advantageously target the destruction of cancer cells while minimizingor eliminating the destruction of normal, non-cancerous cells, therebyreducing, for example, by minimizing or eliminating, toxicity. Moreover,the inventive TCRs (including functional portions and functionalvariants thereof) may, advantageously, successfully treat or preventHPV-positive cancers that do not respond to other types of treatmentsuch as, for example, chemotherapy alone, surgery, or radiation.Additionally, the inventive TCRs (including functional portions andfunctional variants thereof) provide highly avid recognition of HPV 16E7, which may, advantageously, provide the ability to recognizeunmanipulated tumor cells (e.g., tumor cells that have not been treatedwith interferon (IFN)-γ, transfected with a vector encoding one or bothof HPV 16 E7 and HLA-A2, pulsed with the E7₁₁₋₁₉ peptide, or acombination thereof).

The phrase “antigenic specificity,” as used herein, means that the TCR(including functional portions and functional variants thereof) canspecifically bind to and immunologically recognize HPV 16 E7 with highavidity. For example, a TCR (including functional portions andfunctional variants thereof) may be considered to have “antigenicspecificity” for HPV 16 E7 if T cells expressing the TCR (or functionalportion or functional variant thereof) secrete at least about 200 pg/mLor more (e.g., 200 pg/mL or more, 300 pg/mL or more, 400 pg/mL or more,500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more, 1000 pg/mL ormore, 5,000 pg/mL or more, 7,000 pg/mL or more, 10,000 pg/mL or more, or20,000 pg/mL or more) of IFN-γ upon co-culture with antigen-negativeHLA-A2⁺ target cells pulsed with a low concentration of HPV 16 E7peptide (e.g., about 0.05 ng/mL to about 5 ng/mL, 0.05 ng/mL, 0.1 ng/mL,0.5 ng/mL, 1 ng/mL, or 5 ng/mL). Alternatively or additionally, a TCR(including functional portions and functional variants thereof) may beconsidered to have “antigenic specificity” for HPV 16 E7 if T cellsexpressing the TCR (or functional portion or functional variant thereof)secrete at least twice as much IFN-γ as the untransduced peripheralblood lymphocyte (PBL) background level of IFN-γ upon co-culture withantigen-negative HLA-A2+ target cells pulsed with a low concentration ofHPV 16 E7 peptide. Cells expressing the inventive TCRs (includingfunctional portions and functional variants thereof) may also secreteIFN-γ upon co-culture with antigen-negative HLA-A2⁺ target cells pulsedwith higher concentrations of HPV 16 E7 peptide.

The invention provides a TCR comprising two polypeptides (i.e.,polypeptide chains), such as an alpha (u) chain of a TCR, a beta (β)chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR,or a combination thereof. The polypeptides of the inventive TCR cancomprise any amino acid sequence, provided that the TCR has antigenicspecificity for HPV 16 E7.

In an embodiment of the invention, the TCR comprises two polypeptidechains, each of which comprises a human variable region comprising acomplementarity determining region (CDR)1, a CDR2, and a CDR3 of a TCR.In an embodiment of the invention, the TCR comprises a first polypeptidechain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:3 (CDR1 of a chain), a CDR2 comprising the amino acid sequence of SEQ IDNO: 4 (CDR2 of a chain), and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 5 (CDR3 of a chain), and a second polypeptide chaincomprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6(CDR1 of R chain), a CDR2 comprising the amino acid sequence of SEQ IDNO: 7 (CDR2 of R chain), and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 8 (CDR3 of R chain). In this regard, the inventive TCR cancomprise any one or more of the amino acid sequences selected from thegroup consisting of SEQ ID NOs: 3-8. Preferably, the TCR comprises SEQID NOs: 3-5 or SEQ ID NOs: 6-8. In an especially preferred embodiment,the TCR comprises the amino acid sequences of SEQ ID NOs: 3-8.

In an embodiment of the invention, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 9 (the variable region of a human α chain); SEQ ID NO: 10,wherein X at position 2 of SEQ ID NO: 10 is Ala or Gly (the variableregion of a β chain); both SEQ ID NOs: 9 and 10, wherein X at position 2of SEQ ID NO: 10 is Ala or Gly; SEQ ID NO: 11 (the variable region of ahuman β chain); or both SEQ ID NOs: 9 and 11. SEQ ID NO: 10 correspondsto SEQ ID NO: 11 when X at position 2 of SEQ ID NO: 10 is Gly.Preferably, the inventive TCR comprises the amino acid sequences of bothSEQ ID NOs: 9 and 10, wherein X at position 2 of SEQ ID NO: 10 is Ala.

The inventive TCRs may further comprise a constant region derived fromany suitable species such as, e.g., human or mouse. In an embodiment ofthe invention, the TCRs further comprise a human constant region. Inthis regard, the TCR can comprise the amino acid sequence of SEQ ID NO:14 (the constant region of a human α chain), SEQ ID NO: 15 (the constantregion of a human β chain), or both SEQ ID NOs: 14 and 15.

In an embodiment of the invention, the inventive TCR may comprise acombination of a variable region and a constant region. In this regard,the TCR can comprise an alpha chain comprising the amino acid sequencesof both of SEQ ID NO: 9 (the variable region of a human α chain) and SEQID NO: 14 (the constant region of a human α chain); a beta chaincomprising the amino acid sequences of both of SEQ ID NO: 11 (thevariable region of a human β chain) and SEQ ID NO: 15 (the constantregion of a human β chain); a beta chain comprising the amino acidsequences of both of SEQ ID NO: 10, wherein X at position 2 of SEQ IDNO: 2 is Ala or Gly (the variable region of a R chain) and SEQ ID NO: 15(the constant region of a human β chain); the amino acid sequences ofall of SEQ ID NOs: 9, 11, 14, and 15; or the amino acid sequences of allof SEQ ID NOs: 9, 10, 14, and 15.

In an embodiment of the invention, the inventive TCR can comprise an αchain of a TCR and a β chain of a TCR. Each of the α chain and β chainof the inventive TCR can independently comprise any amino acid sequence.In this regard, the α chain of the inventive TCR can comprise the aminoacid sequence of SEQ ID NO: 12. An a chain of this type can be pairedwith any β chain of a TCR. In this regard, the β chain of the inventiveTCR can comprise the amino acid sequence of SEQ ID NO: 13. The inventiveTCR, therefore, can comprise the amino acid sequence of SEQ ID NO: 12,SEQ ID NO: 13, or both SEQ ID NOs: 12 and 13. Preferably, the inventiveTCR is a human TCR comprising the amino acid sequences of both SEQ IDNOs: 12 and 13.

Another embodiment of the invention provides a chimeric TCR comprising ahuman variable region and a murine constant region, or a functionalvariant of the TCR, wherein the TCR and the functional variant haveantigenic specificity for human papillomavirus (HPV) 16 E7. The chimericTCR, or functional variant thereof, may comprise any of the CDR regionsas described herein with respect to other aspects of the invention. Inanother embodiment of the invention, the chimeric TCR, or functionalvariant thereof, may comprise any of the variable regions describedherein with respect to other aspects of the invention.

As used herein, the term “murine” or “human,” when referring to a TCR orany component of a TCR described herein (e.g., complementaritydetermining region (CDR), variable region, constant region, alpha chain,and/or beta chain), means a TCR (or component thereof) which is derivedfrom a mouse or a human, respectively, i.e., a TCR (or componentthereof) that originated from or was, at one time, expressed by a mouseT cell or a human T cell, respectively.

In an embodiment of the invention, the inventive chimeric TCRs comprisea murine constant region. In this regard, the TCR can comprise the aminoacid sequence of SEQ ID NO: 17 (the constant region of a murine (achain), SEQ ID NO: 19 (the constant region of a murine 3 chain), or bothSEQ ID NOs: 17 and 19. In a preferred embodiment, the inventive TCRs arechimeric TCRs comprising both a human variable region and a murineconstant region.

In an embodiment of the invention, the inventive chimeric TCR maycomprise a combination of a variable region and a constant region. Inthis regard, the TCR can comprise an alpha chain comprising the aminoacid sequences of both of SEQ ID NO: 9 (the variable region of a human αchain) and SEQ ID NO: 17 (the constant region of a murine α chain); abeta chain comprising the amino acid sequences of both of SEQ ID NO: 11(the variable region of a human β chain) and SEQ ID NO: 19 (the constantregion of a murine β chain); a beta chain comprising the amino acidsequences of both of SEQ ID NO: 10, wherein X at position 2 of SEQ IDNO: 10 is Ala or Gly (the variable region of a β chain) and SEQ ID NO:19 (the constant region of a murine β chain); the amino acid sequencesof all of SEQ ID NOs: 9, 11, 17, and 19; or the amino acid sequences ofall of SEQ ID NOs: 9, 10, 17, and 19. In an embodiment, the inventivechimeric TCR comprises a full-length beta chain comprising SEQ ID NO:20. In this regard, the TCR can comprise all of SEQ ID NOs: 9, 17, and20.

Included in the scope of the invention are functional variants of theinventive TCRs described herein. The term “functional variant,” as usedherein, refers to a TCR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent TCR,polypeptide, or protein, which functional variant retains the biologicalactivity of the TCR, polypeptide, or protein of which it is a variant.Functional variants encompass, for example, those variants of the TCR,polypeptide, or protein described herein (the parent TCR, polypeptide,or protein) that retain the ability to specifically bind to HPV 16 E7for which the parent TCR has antigenic specificity or to which theparent polypeptide or protein specifically binds, to a similar extent,the same extent, or to a higher extent, as the parent TCR, polypeptide,or protein. In reference to the parent TCR, polypeptide, or protein, thefunctional variant can, for instance, be at least about 30%, 50%, 75%,80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acidsequence to the parent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

In an embodiment of the invention, the functional variant comprises theamino acid sequence of any of the TCRs described herein with one, two,three, or four amino acid substitution(s) in the constant region of thealpha or beta chain. Preferably, the functional variant comprises theamino acid sequence of any of the murine constant regions describedherein with one, two, three, or four amino acid substitution(s) in themurine constant region. In some embodiments, the TCRs (or functionalportions thereof) comprising the substituted amino acid sequence(s)advantageously provide one or more of increased recognition of HPV 16E7V targets, increased expression by a host cell, and increasedanti-tumor activity as compared to the parent TCR comprising anunsubstituted amino acid sequence. In general, the substituted aminoacid sequences of the murine constant regions of the TCR u and R chains,SEQ ID NOs: 16 and 18, respectively, correspond with all or portions ofthe unsubstituted murine constant region amino acid sequences SEQ IDNOs: 17 and 19, respectively, with SEQ ID NO: 16 having one, two, three,or four amino acid substitution(s) when compared to SEQ ID NO: 17 andSEQ ID NO: 18 having one amino acid substitution when compared to SEQ IDNO: 19. In this regard, an embodiment of the invention provides afunctional variant of a TCR comprising the amino acid sequences of (a)SEQ ID NO: 16 (constant region of alpha chain), wherein (i) X atposition 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val,Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly,Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 isGly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 18(constant region of beta chain), wherein X at position 56 is Ser or Cys.In an embodiment of the invention, the functional variant of the TCRcomprising SEQ ID NO: 16 does not comprise SEQ ID NO: 17 (unsubstitutedmurine constant region of alpha chain). In an embodiment of theinvention, the functional variant of the TCR comprising SEQ ID NO: 18does not comprise SEQ ID NO: 19 (unsubstituted murine constant region ofbeta chain).

In an embodiment of the invention, the substituted amino acid sequenceincludes cysteine substitutions in the constant region of one or both ofthe α and β chains to provide a cysteine-substituted TCR. Opposingcysteines in the α and the β chains provide a disulfide bond that linksthe constant regions of the α and the β chains of the substituted TCR toone another and which is not present in a TCR comprising theunsubstituted human constant region or the unsubstituted murine constantregion. In this regard, the functional variant of the TCR is acysteine-substituted, chimeric TCR in which one or both of the nativeThr48 of SEQ ID NO: 17 and the native Ser56 of SEQ ID NO: 19 may besubstituted with Cys. Preferably, both of the native Thr48 of SEQ ID NO:17 and the native Ser56 of SEQ ID NO: 19 are substituted with Cys. In anembodiment, the cysteine-substituted, chimeric TCR comprises an alphachain constant region comprising the amino acid sequence of SEQ ID NO:16, wherein X at position 48 is Cys, X at position 112 is the nativeSer, X at position 114 is the native Met, and X at position 115 is thenative Gly, and a beta chain constant region comprising the amino acidsequence of SEQ ID NO: 18, wherein X at position 56 is Cys. Preferably,the cysteine-substituted, chimeric TCR comprises an alpha chain constantregion comprising the amino acid sequence of SEQ ID NO: 24 and a betachain constant region comprising the amino acid sequence of SEQ ID NO:23. The cysteine-substituted, chimeric TCRs of the invention may includethe substituted constant region in addition to any of the CDRs and/orvariable regions described herein. In this regard, thecysteine-substituted, chimeric TCR can comprise the amino acid sequencesof (i) SEQ ID NOs: 3-5 and 24; (ii) SEQ ID NO: 9 and 24; (iii) SEQ IDNOs: 6-8 and 23; (iv) SEQ ID NOs: 10 and 23, wherein X at position 2 ofSEQ ID NO: 10 is Ala or Gly; (v) SEQ ID NO: 11 and 23; (vi) SEQ ID NOs:9 and 16; (vii) SEQ ID NOs: 10 and 18; (viii) SEQ ID NOs: 11 and 18;(ix) SEQ ID NOs: 3-5 and 16; or (x) SEQ ID NOs: 6-8 and 18. Preferably,the cysteine-substituted, chimeric TCR comprises the amino acidsequences of (i) SEQ ID NOs: 3-8 and 23-24; (ii) SEQ ID NOs: 9-10 and23-24; (iii) SEQ ID NOs: 9, 11, and 23-24; (iv) SEQ ID NOs: 3-8, 16, and18; (v) SEQ ID NOs: 9-10, 16, and 18; or (vi) SEQ ID NOs: 9, 11, 16, and18. In an embodiment, the Cys-substituted, chimeric TCR comprises afull-length beta chain comprising SEQ ID NO: 27. In this regard, theCys-substituted, chimeric TCR can comprise SEQ ID NOs: 9 and 24; SEQ IDNO: 27; or all of SEQ ID NOs: 9, 24, and 27.

In an embodiment of the invention, the substituted amino acid sequenceincludes substitutions of one, two, or three amino acids in thetransmembrane (TM) domain of the constant region of one or both of the αand β chains with a hydrophobic amino acid to provide a hydrophobicamino acid-substituted TCR (also referred to herein as an “LVL-modifiedTCR”). The hydrophobic amino acid substitution(s) in the TM domain ofthe TCR may increase the hydrophobicity of the TM domain of the TCR ascompared to a TCR that lacks the hydrophobic amino acid substitution(s)in the TM domain. In this regard, the functional variant of the TCR isan LVL-modified chimeric TCR in which one, two, or three of the nativeSer112, Met114, and Gly115 of SEQ ID NO: 17 may, independently, besubstituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;preferably with Leu, Ile, or Val. Preferably, all three of the nativeSer112, Met114, and Gly115 of SEQ ID NO: 17 may, independently, besubstituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;preferably with Leu, Ile, or Val. In an embodiment, the LVL-modifiedchimeric TCR comprises an alpha chain constant region comprising theamino acid sequence of SEQ ID NO: 16, wherein X at position 48 is thenative Thr, X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe,Met, or Trp, X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro,Phe, Met, or Trp, and X at position 115 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Met, or Trp, and a beta chain constant region comprising the aminoacid sequence of SEQ ID NO: 19, wherein the LVL-modified chimeric TCRcomprising SEQ ID NO: 16 does not comprise SEQ ID NO: 17 (unsubstitutedmurine constant region of alpha chain). Preferably, the LVL-modified,chimeric TCR comprises an alpha chain constant region comprising theamino acid sequence of SEQ ID NO: 21 and a beta chain constant regioncomprising the amino acid sequence of SEQ ID NO: 19. The LVL-modified,chimeric TCRs of the invention may include the substituted constantregion in addition to any of the CDRs and/or variable regions describedherein. In this regard, the LVL-modified, chimeric TCR can comprise (i)SEQ ID NOs: 3-5 and 21; (ii) SEQ ID NOs: 9 and 21; (iii) SEQ ID NOs: 6-8and 19; (iv) SEQ ID NOs: 10 and 19, wherein X at position 2 of SEQ IDNOs: 10 is Ala or Gly; (v) SEQ ID NOs: 11 and 19; (vi) SEQ ID NOs: 9 and16; (vii) SEQ ID NOs: 3-5 and 16; (x) SEQ ID NOs: 6-8 and 18; (viii) SEQID NO: 10 and 18; or (ix) SEQ ID NO: 11 and 18. Preferably, thecysteine-substituted, chimeric TCR comprises the amino acid sequences of(i) SEQ ID NOs: 3-8 and 19 and 21; (ii) SEQ ID NOs: 9-10 and 19 and 21;(iii) SEQ ID NOs: 9, 11, and 19 and 21; (iv) SEQ ID NOs: 3-8, 16, and18; (v) SEQ ID NOs: 9-10, 16, and 18; or (vi) SEQ ID NOs: 9, 11, 16, and18. In an embodiment, the LVL-modified, chimeric TCR comprises afull-length alpha chain comprising the amino acid sequence of SEQ ID NO:22 and a full-length beta chain comprising the amino acid sequence ofSEQ ID NO: 20. In this regard, the LVL-modified, chimeric TCR cancomprise SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 29, or both SEQ IDNOs: 20 and 22.

In an embodiment of the invention, the substituted amino acid sequenceincludes the cysteine substitutions in the constant region of one orboth of the α and β chains in combination with the substitution(s) ofone, two, or three amino acids in the transmembrane (TM) domain of theconstant region of one or both of the α and β chains with a hydrophobicamino acid (also referred to herein as “cysteine-substituted,LVL-modified TCR”). In this regard, the functional variant of the TCR isa cysteine-substituted, LVL-modified, chimeric TCR in which the nativeThr48 of SEQ ID NO: 17 is substituted with Cys; one, two, or three ofthe native Ser112, Met114, and Gly115 of SEQ ID NO: 17 are,independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met,or Trp; preferably with Leu, Ile, or Val; and the native Ser56 of SEQ IDNO: 19 is substituted with Cys. Preferably, all three of the nativeSer112, Met114, and Gly115 of SEQ ID NO: 17 may, independently, besubstituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;preferably with Leu, Ile, or Val. In an embodiment, thecysteine-substituted, LVL-modified, chimeric TCR comprises an alphachain constant region comprising the amino acid sequence of SEQ ID NO:16, wherein X at position 48 is Cys, X at position 112 is Ser, Gly, Ala,Val, Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 is Met, Gly,Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and X at position 115 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and a beta chain constantregion comprising the amino acid sequence of SEQ ID NO: 18, wherein X atposition 56 is Cys, wherein the cysteine-substituted, LVL-modifiedchimeric TCR comprising SEQ ID NO: 16 does not comprise SEQ ID NO: 17(unsubstituted murine constant region of alpha chain). Preferably, thecysteine-substituted, LVL-modified, chimeric TCR comprises an alphachain constant region comprising the amino acid sequence of SEQ ID NO:25 and a beta chain constant region comprising the amino acid sequenceof SEQ ID NO: 23. The cysteine-substituted, LVL-modified, chimeric TCRsof the invention may include the substituted constant region in additionto any of the CDRs and/or variable regions described herein. In thisregard, the cysteine-substituted, LVL-modified, chimeric TCR cancomprise (i) SEQ ID NOs: 3-5 and 25; (ii) SEQ ID NO: 9 and 25; (iii) SEQID NOs: 6-8 and 23; (iv) SEQ ID NO: 10 and 23, wherein X at position 2of SEQ ID NO: 10 is Ala or Gly; (v) SEQ ID NO: 11 and 23; (vi) SEQ IDNO: 3-5 and 16; (vii) SEQ ID NOs: 9 and 16; (viii) SEQ ID NOs: 6-8 and18; (ix) SEQ ID NOs: 10 and 18; or (x) SEQ ID NOs: 11 and 18.Preferably, the cysteine-substituted, LVL-modified, chimeric TCRcomprises the amino acid sequences of (i) SEQ ID NOs: 3-8 and 23 and 25;(ii) SEQ ID NOs: 9-10 and 23 and 25; (iii) SEQ ID NOs: 9, 11, and 23 and25; (iv) SEQ ID NOs: 3-8, 16, and 18; (v) SEQ ID NOs: 9, 10, 16, and 18;or SEQ ID NOs: 9, 11, 16, and 18. In an especially preferred embodiment,the cysteine-substituted, LVL-modified, chimeric TCR comprises afull-length alpha chain comprising the amino acid sequence of SEQ ID NO:26 and a full-length beta chain comprising the amino acid sequence ofSEQ ID NO: 27. In this regard, the Cys-substituted, LVL-modified,chimeric TCR can comprise SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30,or both SEQ ID NOs: 26 and 27.

The TCR (or functional variant thereof), polypeptide, or protein canconsist essentially of the specified amino acid sequence or sequencesdescribed herein, such that other components of the TCR (or functionalvariant thereof), polypeptide, or protein, e.g., other amino acids, donot materially change the biological activity of the TCR (or functionalvariant thereof), polypeptide, or protein. In this regard, the inventiveTCR (or functional variant thereof), polypeptide, or protein can, forexample, consist essentially of the amino acid sequence of any one ofSEQ ID NOs: 12, 13, 20, 22, 26, 27, 29, and 30. Also, for instance, theinventive TCRs (including functional variants thereof), polypeptides, orproteins can consist essentially of the amino acid sequence(s) of SEQ IDNO: 9, 10, 11, 14-19, 21, 23-25, both SEQ ID NOs: 9 and 10, both SEQ IDNOs: 9 and 11, both SEQ ID NOs: 14 and 15, both SEQ ID NOs: 16 and 18,both SEQ ID NOs: 17 and 19, both SEQ ID NOs: 23 and 24, both SEQ ID NOs:19 and 21, or both SEQ ID NOs: 23 and 25. Furthermore, the inventiveTCRs (including functional variants thereof), polypeptides, or proteinscan consist essentially of the amino acid sequence of SEQ ID NO: 3 (CDR1of a chain), SEQ ID NO: 4 (CDR2 of a chain), SEQ ID NO: 5 (CDR3 of achain), SEQ ID NO: 6 (CDR1 of R chain), SEQ ID NO: 7 (CDR2 of R chain),SEQ ID NO: 8 (CDR3 of R chain), or any combination thereof, e.g., SEQ IDNOs: 3-5; 6-8; or 3-8.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein. The term “polypeptide” as used herein includes oligopeptides andrefers to a single chain of amino acids connected by one or more peptidebonds.

With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR (orfunctional variant thereof) of which it is a part, provided that thefunctional portion specifically binds to HPV 16 E7. The term “functionalportion” when used in reference to a TCR (or functional variant thereof)refers to any part or fragment of the TCR (or functional variantthereof) of the invention, which part or fragment retains the biologicalactivity of the TCR (or functional variant thereof) of which it is apart (the parent TCR or parent functional variant thereof). Functionalportions encompass, for example, those parts of a TCR (or functionalvariant thereof) that retain the ability to specifically bind to HPV 16E7 (e.g., in an HLA-A2-dependent manner), or detect, treat, or preventcancer, to a similar extent, the same extent, or to a higher extent, asthe parent TCR (or functional variant thereof). In reference to theparent TCR (or functional variant thereof), the functional portion cancomprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, ormore, of the parent TCR (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCRor functional variant thereof. Desirably, the additional amino acids donot interfere with the biological function of the functional portion,e.g., specifically binding to HPV 16 E7; and/or having the ability todetect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent TCR or functional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and CDR3 of the variable region(s) of the α chain and/or β chainof a TCR or functional variant thereof of the invention. In anembodiment of the invention, the polypeptide can comprise a functionalportion comprising the amino acid sequence of SEQ ID NO: 3 (CDR1 of αchain), 4 (CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of β chain), 7(CDR2 of β chain), 8 (CDR3 of β chain), or a combination thereof.Preferably, the inventive polypeptide comprises a functional portioncomprising SEQ ID NOs: 3-5; 6-8; or all of SEQ ID NOs: 3-8. Morepreferably, the polypeptide comprises a functional portion comprisingthe amino acid sequences of all of SEQ ID NOs: 3-8.

In an embodiment of the invention, the inventive polypeptide cancomprise, for instance, the variable region of the inventive TCR orfunctional variant thereof comprising a combination of the CDR regionsset forth above. In this regard, the polypeptide can comprise the aminoacid sequence of SEQ ID NO: 9 (the variable region of an α chain), SEQID NO: 10, wherein X at position 2 of SEQ ID NO: 10 is Ala or Gly (thevariable region of a R chain), SEQ ID NO: 11 (the variable region of a Rchain), both SEQ ID NOs: 9 and 10, or both SEQ ID NOs: 9 and 11.Preferably, the polypeptide comprises the amino acid sequences of bothSEQ ID NOs: 9 and 10, wherein X at position 2 of SEQ ID NO: 10 is Ala.

The inventive polypeptide may further comprise a constant region derivedfrom any suitable species such as, e.g., human or mouse, describedherein or any of the substituted constant regions described herein. Inthis regard, the polypeptide can comprise the amino acid sequence of SEQID NO: 14 (the constant region of a human α chain), SEQ ID NO: 15 (theconstant region of a human β chain), SEQ ID NO: 16 (constant region of αchain, substituted as described herein with respect to other aspects ofthe invention), SEQ ID NO: 17 (the constant region of a murine α chain),SEQ ID NO: 18 (constant region of R chain, substituted as describedherein with respect to other aspects of the invention), SEQ ID NO: 19(the constant region of a murine R chain), SEQ ID NO: 21 (constantregion of an LVL-modified α chain), SEQ ID NO: 23 (constant region of aCys-substituted R chain), SEQ ID NO: 24 (constant region of aCys-substituted α chain), SEQ ID NO: 25 (constant region of aCys-substituted, LVL-modified α chain), both SEQ ID NOs: 14 and 15, bothSEQ ID NOs: 16 and 18, both SEQ ID NOs: 17 and 19, both SEQ ID NOs: 19and 21, both SEQ ID NOs: 23 and 24, or both SEQ ID NOs: 23 and 25.Preferably, the polypeptide comprises both SEQ ID NOs: 14 and 15, bothSEQ ID NOs: 16 and 18, both SEQ ID NOs: 17 and 19, both SEQ ID NOs: 19and 21, both SEQ ID NOs: 23 and 24, or both SEQ ID NOs: 23 and 25. In anembodiment, the polypeptide comprising the amino acid sequence of one orboth of SEQ ID NOs: 9 and 11 is isolated or purified.

In an embodiment of the invention, the inventive polypeptide maycomprise a combination of a variable region and a constant region. Inthis regard, the polypeptide can comprise both SEQ ID NOs: 9 and 14,both SEQ ID NOs: 9 and 16, both SEQ ID NOs: 9 and 17, both SEQ ID NOs: 9and 21, both SEQ ID NOs: 9 and 24, both SEQ ID NOs: 9 and 25, both SEQID NOs: 10 and 15, both SEQ ID NOs: 10 and 18, both SEQ ID NOs: 10 and19, both SEQ ID NOs: 10 and 23, both SEQ ID NOs: 11 and 15, both SEQ IDNOs: 11 and 18, both SEQ ID NOs: 11 and 19, both SEQ ID NOs: 11 and 23,all of SEQ ID NOs: 3-5 and 14, all of SEQ ID NOs: 3-5 and 16, all of SEQID NOs: 3-5 and 17, all of SEQ ID NOs: 3-5 and 21, all of SEQ ID NOs:3-5 and 24, all of SEQ ID NOs: 3-5 and 25, all of SEQ ID NOs: 6-8 and15, all of SEQ ID NOs: 6-8 and 18, all of SEQ ID NOs: 6-8 and 19, all ofSEQ ID NOs: 6-8 and 23, all of SEQ ID NOs: 3-8 and 14-15, all of SEQ IDNOs: 3-8 and 16 and 18, all of SEQ ID NOs: 3-8 and 17 and 19, all of SEQID NOs: 3-8 and 19 and 21, all of SEQ ID NOs: 3-8 and 23 and 24, all ofSEQ ID NOs: 3-8 and 23 and 25, all of SEQ ID NOs: 9-10 and 14-15, all ofSEQ ID NOs: 9-10 and 16 and 18, all of SEQ ID NOs: 9-10 and 17 and 19,all of SEQ ID NOs: 9-10 and 19 and 21, all of SEQ ID NOs: 9-10 and 23and 24, all of SEQ ID NOs: 9-10 and 23 and 25, all of SEQ ID NOs: 9, 11,and 14-15, all of SEQ ID NOs: 9, 11, and 16 and 18, all of SEQ ID NOs:9, 11, and 17 and 19, all of SEQ ID NOs: 9, 11, and 19 and 21, all ofSEQ ID NOs: 9, 11, and 23 and 24, or all of SEQ ID NOs: 9, 11, and 23and 25. SEQ ID NOs: 16 and 18 may be substituted as described hereinwith respect to other aspects of the invention. In an embodiment, thepolypeptide comprising the amino acid sequence of one or both of (i) SEQID NOs: 9 and 14 and (ii) SEQ ID NOs: 11 and 15 is isolated or purified.

In an embodiment of the invention, the inventive polypeptide cancomprise the entire length of an a or β chain of one of the TCRs orfunctional variant thereof described herein. In this regard, theinventive polypeptide can comprise an amino acid sequence of (i) any oneof SEQ ID NOs: 12, 13, 20, 22, 26, 27, 29, 30; (ii) SEQ ID NOs: 9, 24,and 27; (iii) SEQ ID NOs: 9, 17, and 20; or (iv) SEQ ID NOs: 9, 10, 16,and 18. SEQ ID NOs: 16 and 18 may be substituted as described hereinwith respect to other aspects of the invention.

Alternatively, the polypeptide of the invention can comprise α and βchains of the TCRs or functional variants thereof described herein. Forexample, the inventive polypeptide can comprise the amino acid sequencesof both SEQ ID NOs: 12 and 13; both SEQ ID NOs: 20 and 22; both SEQ IDNOs: 26 and 27; all of SEQ ID NOs: 9, 24, and 27; all of SEQ ID NOs: 9,17, and 20; or all of SEQ ID NOs: 9, 10, 16, and 18. Preferably, thepolypeptide comprises the amino acid sequences of both SEQ ID NOs: 12and 13; both SEQ ID NOs: 20 and 22; both SEQ ID NOs: 26 and 27; all ofSEQ ID NOs: 9, 24, and 27; all of SEQ ID NOs: 9, 17, and 20; or all ofSEQ ID NOs: 9, 10, 16, and 18. SEQ ID NOs: 16 and 18 may be substitutedas described herein with respect to other aspects of the invention. Inan embodiment, the polypeptide comprising the amino acid sequence of oneor both of SEQ ID NOs: 12 and 13 is isolated or purified.

The invention further provides a protein comprising at least one of thepolypeptides described herein. By “protein” is meant a moleculecomprising one or more polypeptide chains. In an embodiment, the proteincomprising (a) one or both of the amino acid sequences of SEQ ID NO: 9and SEQ ID NO: 10, wherein X at position 2 of SEQ ID NO: 10 is Gly, or(b) one or both of SEQ ID NO: 12 and 13 is isolated or purified.

In an embodiment, the protein of the invention can comprise a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs: 3-5and a second polypeptide chain comprising the amino acid sequence of SEQID NOs: 6-8. Alternatively or additionally, the protein of the inventioncan comprise a first polypeptide chain comprising the amino acidsequence of SEQ ID NO: 9 and a second polypeptide chain comprising theamino acid sequence of SEQ ID NO: 10, wherein (i) X at position 2 of SEQID NO: 10 is Ala or Gly, and (ii) the protein comprising SEQ ID NOs: 9and 10, wherein X at position 2 of SEQ ID NO: 10 is Gly, is isolated orpurified. The protein can, for example, comprise a first polypeptidechain comprising the amino acid sequence of (i) SEQ ID NO: 12, (ii) SEQID NO: 22, (iii) SEQ ID NO: 26, (iv) SEQ ID NO: 9 and 16, (v) SEQ ID NO:9 and 17, or (vi) SEQ ID NO: 9 and 24 and a second polypeptide chaincomprising the amino acid sequence of (i) SEQ ID NO: 10 and 18, or (ii)any one of SEQ ID NOs: 13, 20, and 27, wherein the protein comprisingSEQ ID NO: 12 and 13 is isolated or purified, and SEQ ID NOs: 16 and 18are substituted as described herein with respect to other aspects of theinvention. In this instance, the protein of the invention can be a TCR.Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising both SEQ ID NOs: 12 and 13, both SEQ ID NO:20 and 22, SEQ ID NO: 26 and 27, all of SEQ ID NOs: 9, 10, 16, and 18,all of SEQ ID NOs: 9, 17, and 20, all of SEQ ID NOs: 9, 24, and 27, orif the first and/or second polypeptide chain(s) of the protein furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide can encode any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

In some embodiments of the invention, the TCRs (and functional portionsand functional variants thereof), polypeptides, and proteins of theinvention may be expressed as a single protein comprising a linkerpeptide linking the α chain and the β chain. In this regard, the TCRs(and functional variants and functional portions thereof), polypeptides,and proteins of the invention comprising both SEQ ID NOs: 12 and 13,both SEQ ID NO: 20 and 22, SEQ ID NO: 26 and 27, all of SEQ ID NOs: 9,10, 16, and 18, all of SEQ ID NOs: 9, 17, and 20, all of SEQ ID NOs: 9,24, and 27 may further comprise a linker peptide. The linker peptide mayadvantageously facilitate the expression of a recombinant TCR (includingfunctional portions and functional variants thereof), polypeptide,and/or protein in a host cell. The linker peptide may comprise anysuitable amino acid sequence. For example, the linker peptide maycomprise SEQ ID NO: 28. In an embodiment of the invention, the proteincomprising an alpha chain, beta chain, and a linker may comprise SEQ IDNO: 29 (LVL-modified, chimeric TCR) or SEQ ID NO: 30 (Cys-substituted,LVL-modified, chimeric TCR). Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may be cleaved,resulting in separated a and β chains.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof,can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment, including any of theantibodies and antibody fragments described herein.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be of any length, i.e., can compriseany number of amino acids, provided that the TCRs, polypeptides, orproteins (or functional variants thereof) retain their biologicalactivity, e.g., the ability to specifically bind to HPV 16 E7; detectcancer, HPV 16 infection, or HPV-positive premalignancy in a mammal; ortreat or prevent cancer, HPV 16 infection, or HPV-positive premalignancyin a mammal, etc. For example, the polypeptide can be in the range offrom about 50 to about 5000 amino acids long, such as 50, 70, 75, 100,125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or moreamino acids in length. In this regard, the polypeptides of the inventionalso include oligopeptides.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) of the invention can comprise syntheticamino acids in place of one or more naturally-occurring amino acids.Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, omithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

The TCR, polypeptide, and/or protein of the invention (includingfunctional variants thereof) can be obtained by methods known in theart. Suitable methods of de novo synthesizing polypeptides and proteinsare described in references, such as Chan et al., Fmoc Solid PhasePeptide Synthesis, Oxford University Press, Oxford, United Kingdom,2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford UniversityPress, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also,polypeptides and proteins can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Green and Sambrook, Molecular Cloning: A LaboratoryManual, 4^(th) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.2012; and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, N Y, 1994. Further, some ofthe TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be isolated and/or purified from asource, such as a plant, a bacterium, an insect, a mammal, e.g., a rat,a human, etc. Methods of isolation and purification are well-known inthe art. Alternatively, the TCRs, polypeptides, and/or proteinsdescribed herein (including functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, Calif.),Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple PeptideSystems (San Diego, Calif.). In this respect, the inventive TCRs(including functional variants thereof), polypeptides, and proteins canbe synthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive TCRs, polypeptides, orproteins (including any of the functional variants thereof), nucleicacids, recombinant expression vectors, host cells, populations of hostcells, or antibodies, or antigen binding portions thereof. Conjugates,as well as methods of synthesizing conjugates in general, are known inthe art (See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223(2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

An embodiment of the invention provides a nucleic acid sequencecomprising a nucleotide sequence encoding any of the TCRs (includingfunctional portions and functional variants thereof, polypeptides, orproteins described herein. By “nucleic acid” as used herein includes“polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” andgenerally means a polymer of DNA or RNA, which can be single-stranded ordouble-stranded, synthesized or obtained (e.g., isolated and/orpurified) from natural sources, which can contain natural, non-naturalor altered nucleotides, and which can contain a natural, non-natural oraltered internucleotide linkage, such as a phosphoroamidate linkage or aphosphorothioate linkage, instead of the phosphodiester found betweenthe nucleotides of an unmodified oligonucleotide. In an embodiment, thenucleic acid comprises complementary DNA (cDNA). It is generallypreferred that the nucleic acid does not comprise any insertions,deletions, inversions, and/or substitutions. However, it may be suitablein some instances, as discussed herein, for the nucleic acid to compriseone or more insertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Green and Sambrook et al., supra, and Ausubel et al., supra.For example, a nucleic acid can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that can be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the TCRs, polypeptides, proteins, or functional variants thereofdescribed herein. In an embodiment of the invention, the nucleotidesequence may comprise, consist, or consist essentially of any one of SEQID NO: 31 (alpha chain of human, wild-type TCR), SEQ ID NO: 32 (betachain of human, wild-type TCR), SEQ ID NO: 33 (alpha chain ofLVL-modified, chimeric TCR), SEQ ID NO: 34 (beta chain of chimeric TCR),SEQ ID NO: 35 (alpha chain of Cys-substituted, LVL-modified, chimericTCR), SEQ ID NO: 36 (beta chain of Cys-substituted, chimeric TCR), bothSEQ ID NOs: 31 and 32, both SEQ ID NOs: 33 and 34, or both SEQ ID NOs:35 and 36.

In an embodiment of the invention, the nucleic acid comprises anon-natural nucleotide sequence. A nucleotide sequence may be consideredto be “non-natural” if the nucleotide sequence is not found in nature.In some embodiments, the nucleotide sequence may be codon-optimized.Without being bound to a particular theory or mechanism, it is believedthat codon optimization of the nucleotide sequence increases thetranslation efficiency of the mRNA transcripts. Codon optimization ofthe nucleotide sequence may involve substituting a native codon foranother codon that encodes the same amino acid, but can be translated bytRNA that is more readily available within a cell, thus increasingtranslation efficiency. Optimization of the nucleotide sequence may alsoreduce secondary mRNA structures that would interfere with translation,thus increasing translation efficiency. In an embodiment of theinvention, the codon-optimized nucleotide sequence may comprise,consist, or consist essentially of any one of SEQ ID NOs: 33-36, both ofSEQ ID NOs: 33 and 34, or both of SEQ ID NOs: 35 and 36.

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs(including functional portions and functional variants thereof). It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. In an embodiment of the invention, the recombinantexpression vector comprises a nucleotide sequence encoding the α chain,the 3 chain, and linker peptide. For example, in an embodiment, therecombinant expression vector comprises a codon-optimized nucleotidesequence comprising SEQ ID NO: 39 (encoding chimeric α and β chains SEQID NOs: 20 and 22 with a linker positioned between them, wherein thenucleotide sequence encoding the beta chain is positioned 5′ of thenucleotide sequence encoding the alpha chain) or SEQ ID NO: 40 (encodingchimeric α and β chains SEQ ID NOs: 26 and 27 with a linker positionedbetween them, wherein the nucleotide sequence encoding the beta chain ispositioned 5′ of the nucleotide sequence encoding the alpha chain).

For purposes herein, the term “recombinant expression vector” means agenetically-modified oligonucleotide or polynucleotide construct thatpermits the expression of an mRNA, protein, polypeptide, or peptide by ahost cell, when the construct comprises a nucleotide sequence encodingthe mRNA, protein, polypeptide, or peptide, and the vector is contactedwith the cell under conditions sufficient to have the mRNA, protein,polypeptide, or peptide expressed within the cell. The vectors of theinvention are not naturally-occurring as a whole. However, parts of thevectors can be naturally-occurring. The inventive recombinant expressionvectors can comprise any type of nucleotides, including, but not limitedto DNA and RNA, which can be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors can comprise naturally-occurring,non-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder the transcription orreplication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector. In an especially preferred embodiment, the recombinantexpression vector is an MSGV1 vector.

In a preferred embodiment, the recombinant expression vector comprises anucleotide sequence encoding an alpha chain and a beta chain of any ofthe TCRs (including functional portions and functional variants thereof)described herein, wherein the nucleotide sequence encoding the betachain is positioned 5′ of the nucleotide sequence encoding the alphachain. In this regard, the nucleotide sequence encoding the alpha chainmay be positioned 3′ of the nucleotide sequence encoding the beta chain.Without being bound by a particular theory or mechanism, it is believedthat a nucleotide sequence encoding a beta chain that is positioned 5′of the nucleotide sequence encoding the alpha chain may provide any oneor more of increased recognition of HPV 16 E7⁺ targets, increasedexpression by a host cell, and increased anti-tumor activity as comparedto a nucleotide sequence encoding a beta chain that is positioned 3′ ofthe nucleotide sequence encoding the alpha chain. In a less preferredembodiment, the nucleotide sequence encoding the beta chain ispositioned 3′ of the nucleotide sequence encoding the alpha chain. Inthis regard, the nucleotide sequence encoding the alpha chain may bepositioned 5′ of the nucleotide sequence encoding the beta chain. In anembodiment, an MSGV1 vector comprising a codon-optimized nucleotidesequence encoding an LVL-modified, chimeric TCR comprising SEQ ID NOs:20 and 22 of the invention, wherein the nucleotide sequence encoding thebeta chain is positioned 5′ of the nucleotide sequence encoding thealpha chain, comprises SEQ ID NO: 37. In another embodiment, an MSGV1vector comprising a codon-optimized nucleotide sequence encoding aCys-substituted, LVL-modified, chimeric TCR comprising SEQ ID NOs: 26and 27 of the invention, wherein the nucleotide sequence encoding thebeta chain is positioned 5′ of the nucleotide sequence encoding thealpha chain, comprises SEQ ID NO: 38.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Green and Sambrook et al., supra, and Ausubel et al., supra. Constructsof expression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papillomavirus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host cell (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostcell to provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the TCR,polypeptide, or protein (including functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the TCR, polypeptide, or protein(including functional variants thereof). The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5a E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5a cell. For purposes of producing arecombinant TCR, polypeptide, or protein, the host cell is preferably amammalian cell. Most preferably, the host cell is a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell preferablyis a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclearcell (PBMC). More preferably, the host cell is a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. Preferably, the T cell is a human T cell. More preferably,the T cell is a T cell isolated from a human. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells,e.g., Th₁ and Th₂ cells, CD4⁺ T cells, CD8⁺ T cells (e.g., cytotoxic Tcells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g.,central memory T cells and effector memory T cells), naïve T cells, andthe like.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

In an embodiment of the invention, the numbers of cells in thepopulation may be rapidly expanded. Expansion of the numbers of T cellscan be accomplished by any of a number of methods as are known in theart as described in, for example, U.S. Pat. Nos. 8,034,334; 8,383,099;U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J.Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods,128:189-201 (1990).

The invention further provides an antibody, or antigen binding portionthereof, which specifically binds to a functional portion of any of theTCRs (or functional variant thereof) described herein. Preferably, thefunctional portion specifically binds to the cancer antigen, e.g., thefunctional portion comprising the amino acid sequence SEQ ID NO: 3 (CDR1of α chain), 4 (CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of βchain), 7 (CDR2 of β chain), 8 (CDR3 of β chain), SEQ ID NO: 9 (variableregion of α chain), SEQ ID NO: 10 (variable region of R chain), SEQ IDNO: 11 (variable region of R chain), or a combination thereof, e.g.,3-5; 6-8; 3-8; 9; 10; 11; 9-10; or 9 and 11. More preferably, thefunctional portion comprises the amino acid sequences of SEQ ID NOs:3-8; SEQ ID NOs: 9 and 10; or SEQ ID NOs: 9 and 11. In a preferredembodiment, the antibody, or antigen binding portion thereof, binds toan epitope which is formed by all 6 CDRs (CDR1-3 of the alpha chain andCDR1-3 of the beta chain). The antibody can be any type ofimmunoglobulin that is known in the art. For instance, the antibody canbe of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody canbe monoclonal or polyclonal. The antibody can be a naturally-occurringantibody, e.g., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. The antibody can bein monomeric or polymeric form. Also, the antibody can have any level ofaffinity or avidity for the functional portion of the inventive TCR (orfunctional variant thereof). Desirably, the antibody is specific for thefunctional portion of the inventive TCR (or functional variantsthereof), such that there is minimal cross-reaction with other peptidesor proteins.

Methods of testing antibodies for the ability to bind to any functionalportion or functional variant of the inventive TCR are known in the artand include any antibody-antigen binding assay, such as, for example,radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., infra, andU.S. Patent Application Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway etal. (eds.), Immunobiology, 8^(th) Ed., Garland Publishing, New York,N.Y. (2011)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1.

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Green andSambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^(th)Edition, Cold Spring Harbor Laboratory Press, New York (2012)). Phageencoding a variable region with the desired specificity are selected forspecific binding to the desired antigen, and a complete or partialantibody is reconstituted comprising the selected variable domain.Nucleic acid sequences encoding the reconstituted antibody areintroduced into a suitable cell line, such as a myeloma cell used forhybridoma production, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in, for example, U.S. Pat. No. 5,639,641 and Pedersen et al.,J Mol. Biol., 235, 959-973 (1994).

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

The inventive TCRs, polypeptides, proteins, (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), can be isolated and/or purified. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. For example, the purity can beat least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or canbe 100%.

The inventive TCRs, polypeptides, proteins (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), all of which are collectively referred to as“inventive TCR materials” hereinafter, can be formulated into acomposition, such as a pharmaceutical composition. In this regard, theinvention provides a pharmaceutical composition comprising any of theTCRs, polypeptides, proteins, functional portions, functional variants,nucleic acids, expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof)described herein, and a pharmaceutically acceptable carrier. Theinventive pharmaceutical compositions containing any of the inventiveTCR materials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as a chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the particular inventive TCR material underconsideration. Such pharmaceutically acceptable carriers are well-knownto those skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Suitable formulations may include any of those for oral,parenteral, subcutaneous, intravenous, intramuscular, intraarterial,intrathecal, or interperitoneal administration. More than one route canbe used to administer the inventive TCR materials, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered should be sufficient to effect,e.g., a therapeutic or prophylactic response, in the subject or animalover a reasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR (or functional variant or functionalportion thereof), polypeptide, or protein upon administration of a givendose of such T cells to a mammal among a set of mammals of which is eachgiven a different dose of the T cells, could be used to determine astarting dose to be administered to a mammal. The extent to which targetcells are lysed or IFN-γ is secreted upon administration of a certaindose can be assayed by methods known in the art.

The dose of the inventive TCR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive TCR material.Typically, the attending physician will decide the dosage of theinventive TCR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive TCR material to beadministered, route of administration, and the severity of the conditionbeing treated. In an embodiment in which the inventive TCR material is apopulation of cells, the number of cells administered per infusion mayvary, e.g., from about 1×10⁶ to about 1×10¹² cells or more. In certainembodiments, fewer than 1×10⁶ cells may be administered.

One of ordinary skill in the art will readily appreciate that theinventive TCR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive TCR materials is increased through the modification. Forinstance, the inventive TCR materials can be conjugated either directlyor indirectly through a bridge to a targeting moiety. The practice ofconjugating compounds, e.g., inventive TCR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the inventive TCR materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “bridge” as used herein, refers to any agent or molecule thatlinks the inventive TCR materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive TCRmaterials, which are not necessary for the function of the inventive TCRmaterials, are ideal sites for attaching a bridge and/or a targetingmoiety, provided that the bridge and/or targeting moiety, once attachedto the inventive TCR materials, do(es) not interfere with the functionof the inventive TCR materials, i.e., the ability to bind to HPV 16 E7;or to detect, treat, or prevent cancer, HPV 16 infection, orHPV-positive premalignancy.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional variants thereof), polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, or populations ofcells can be used in methods of treating or preventing cancer, HPV 16infection, or HPV-positive premalignancy. Without being bound to aparticular theory, the inventive TCRs (and functional variants thereof)are believed to bind specifically to HPV 16 E7, such that the TCR (orrelated inventive polypeptide or protein and functional variantsthereof), when expressed by a cell, is able to mediate an immuneresponse against a target cell expressing HPV 16 E7. In this regard, theinvention provides a method of treating or preventing a condition in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, TCRs (and functional variants thereof), polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs (andfunctional variants thereof), polypeptides, proteins described herein,or any host cell or population of cells comprising a recombinant vectorwhich encodes any of the TCRs (and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs (and functionalvariants thereof), polypeptides, proteins, nucleic acids, recombinantexpression vectors, host cells, populations of cells, or antibodies, orantigen binding portions thereof, described herein, can be labeled witha detectable label such as, for instance, a radioisotope, a fluorophore(e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme(e.g., alkaline phosphatase, horseradish peroxidase), and elementparticles (e.g., gold particles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer,cancer of the anus, anal canal, or anorectum, cancer of the eye, cancerof the intrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vagina, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, uterine cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is HPV 16-positive cancer. While thecancers most commonly associated with HPV 16 infection include cancer iscancer of the uterine cervix, oropharynx, anus, anal canal, anorectum,vagina, vulva, and penis, the inventive methods may be used to treat anyHPV 16-positive cancer, including those that occur at other anatomicalareas.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the isolation of a human anti-HPV 16 E7 TCRfrom neoplasia.

Samples of lymphocytes from HPV 16-positive cervical intraepithelialneoplasia (CIN) II/III were obtained from fourteen patients. Thepatients had previously received various vaccines targeting HPV 16 E7.Numbers of cervix infiltrating lymphocytes (CIL) were expanded using theRapid Expansion Protocol (REP) as previously described (Dudley et al. JImmunother. 26:332-42 (2003) and Riddell et al. J Immunol. Methods128:189-201 (1990)). Briefly, TIL were cultured with irradiated (40 Gy)allogeneic peripheral blood mononuclear “feeder” cells in completemedium (CM) with 30 ng/mL anti-CD3 antibody and 6000 IU/mL IL-2. Theexpanded numbers of CIL were screened for HPV 16 E7 reactivity bymeasuring interferon (IFN)-γ secretion following co-culture withautologous dendritic cells (DCs) pulsed with gp100 peptide, a pool of15-mer E6 peptides that overlap by 11 amino acid residues and which spanthe whole length of HPV 16 E6, a pool of 15-mer E7 peptides that overlapby 11 amino acid residues and which span the whole length of HPV 16 E7,or OKT3. The CD8⁺ CIL of one patient (5048) were identified as havingHPV 16 E7 reactivity. The patient 5048 was also found to expressHLA-A*02:01.

A tetramer of HLA-A*02:01/E7₁₁₋₁₉ was used to determine the presence ofT cells from patient 5048 targeting the epitope HPV 16 E7₁₁₋₁₉.Tetramer-binding cells were isolated using magnetic bead separationusing anti-PE antibody and limiting dilution cloning was performed. Tcells clones were screened for tetramer binding and a high-binding clonewas identified.

The variable regions of the alpha and beta chains of the TCR of theclone that bound the HLA-A2/E7₁₁₋₁₉ tetramer was isolated and sequencedusing 5′ Rapid Amplification of cDNA Ends (RACE) polymerase chainreaction (PCR). A nucleotide sequence comprising cDNA encoding thevariable region of a wild-type human α chain comprising SEQ ID NO: 9 wasobtained from TRAV1-2*01/TRAJ7*01. A nucleotide sequence comprising cDNAencoding the variable region of a wild-type human β chain comprising SEQID NO: 11 was obtained fromTRBV5-6*01/TRBJ2-1/TRBD2.

Example 2

This example demonstrates that peripheral blood T cells transduced toexpress a chimeric anti-HPV 16 E7 TCR comprising a human variable regionand a mouse constant region displayed CD8-independent binding toHLA-A2/E7₁₁₋₁₉ tetramer and recognized HLA-A2⁺ HPV-16⁺ tumor lines.

An MSGV1 recombinant expression vector comprising nucleotide sequencesencoding a chimeric anti-HPV 16 E7 TCR comprising a human variableregion derived from the wild-type, human TCR of Example 1 and a mouseconstant region was prepared as follows. The nucleotide sequences in therecombinant expression vector encoded the variable region of the α chainand the variable region of the β chain of the TCR of Example 1, with theexception that an alanine was substituted for the native glycine in thesecond position of the variable region of the β chain (the leadersequence) in order to provide a NcoI restriction site and Kozak sequencein the recombinant expression vector. Nucleotide sequences encoding amurine constant region of the α and β chains (SEQ ID NOs: 17 and 19,respectively) were inserted into the vector in place of the respectivehuman constant regions to provide a nucleotide sequence encoding achimeric TCR. A codon-optimized nucleotide sequence encoding apicomavirus 2A peptide (SEQ ID NO: 28) was positioned between the α andβ chains. The nucleotide sequence encoding the chimeric α and β chainsand linker were codon-optimized for expression in human tissues.

Peripheral blood cells were transduced with retrovirus from the MSGV1recombinant expression vector encoding the chimeric TCR or a vectorencoding a fully murine, anti-HPV 16 E7 TCR. Untransduced cells wereused as a negative control. The transduced cells were labeled withanti-CD8 and anti-TRBC (mouse constant region) antibodies and tested forbinding to HLA-A2/E7₁₁₋₁₉ tetramer by flow cytometry. Untransduced cells(both CD8⁺ and CD8⁻) and cells transduced to express a control mouseanti-HPV 16 E7 TCR that was identified in prior experiments (both CD8⁺and CD8⁻) did not bind the tetramer. Both CD8⁺ and CD8⁻ cells transducedwith the chimeric HPV-16 E7 TCR bound the tetramer. Accordingly, PBLtransduced with the chimeric TCR bound HLA-A2/E7₁₁₋₁₉ tetramer in aCD8-independent manner. It was also observed that about 50% of thetransduced T cells expressing the β chain of the chimeric TCR did notbind to tetramer.

In a separate experiment, peripheral blood lymphocytes (PBL) weretransduced with the MSGV1 recombinant expression vector encoding thechimeric TCR with approximately 50% transduction efficiency.HLA-A2/E7₁₁₋₁₉ tetramer binding was measured by fluorescence-activatedcell sorting (FACS), and 25% of the cells were found to bind tetramer onday 8 after stimulation. Transduced cells were co-cultured with target293-A2 cells pulsed with HPV 16 E6₂₉₋₃₈ peptide (control), 293-A2 cellspulsed with HPV 16 E7₁₁₋₁₉ peptide, 624 cells transduced with a plasmidencoding HPV 16 E6, 624 cells transduced with a plasmid encoding HPV 16E7, SCC152 cells, SCC90 cells, CaSki cells, Alb cells, Panc1 cells, orSiHa cells 10 days after stimulation. Untransduced cells were used as acontrol. IFN-γ was measured. The results are shown in FIG. 1. As shownin FIG. 1, PBL transduced with the MSGV1 recombinant expression vectorencoding the chimeric TCR specifically recognized HPV 16 E7-positivetumor cell lines and other HLA-A2⁺HPV16 E7⁺ targets in anHLA-A2-restricted manner. Results obtained with the cells of a seconddonor were similar.

Example 3

This example demonstrates a method of making a chimeric anti-HPV 16 E7TCR comprising a human variable region and a mouse constant region,wherein three native amino acid residues in the transmembrane (TM)region of the constant region of the α chain of the TCR are eachsubstituted with a hydrophobic amino acid residue.

A nucleotide sequence encoding a chimeric TCR comprising a humanvariable region and a mouse constant region, wherein three native aminoacid residues in the transmembrane (TM) region of the constant region ofthe α chain of the TCR are each substituted with a hydrophobic aminoacid residue, was prepared as follows. The nucleotide sequences encodingthe α and β chains of the chimeric TCR of Example 2 were cloned into asingle nucleotide sequence with the nucleotide sequence encoding the βchain positioned 5′ of the nucleotide sequence encoding the alpha chainand a nucleotide sequence encoding a picornavirus 2A peptide (SEQ ID NO:28) positioned between the α and β chains. With reference to thewild-type α chain mouse constant region SEQ ID NO: 17, three nativeamino acid residues in the TM region of the α chain (namely, the Ser,Met, and Gly at positions 112, 114, and 115, respectively, weresubstituted with a Leu, Ile, and Val, respectively. The combinednucleotide sequence was codon-optimized for expression in human tissuesto provide a vector insert (SEQ ID NO: 39). The vector insert was clonedinto an MSGV1 expression vector resulting in SEQ ID NO: 37. The TCRencoded by the vector comprised an α chain comprising an amino acidsequence comprising SEQ ID NO: 22 and a β chain comprising an amino acidsequence comprising SEQ ID NO: 20 (“LVL-modified TCR” or “LVL TCR”).

Example 4

This example demonstrates a method of making chimeric anti-HPV 16 E7TCRs comprising a human variable region and a mouse constant region,wherein a native amino acid residue in the β and α chains are eachsubstituted with a cysteine residue.

The TCR of Example 2 was modified to include a Cys substitution in theconstant region of each of the α and β chains as follows. The nucleotidesequence encoding the constant region of the α chain of the TCR ofExample 2 (amino acid SEQ ID NO: 17) was modified to substitute thenative Thr at position 48 with Cys. The nucleotide sequence encoding theconstant region of the β chain of the TCR of Example 2 (SEQ ID NO: 19)was modified to substitute the native Ser at position 56 with Cys. Thenucleotide sequences encoding the α and β chains were cloned into asingle nucleotide sequence with the nucleotide sequence encoding the βchain positioned 5′ of the nucleotide sequence encoding the α chain anda nucleotide sequence encoding a picomavirus 2A peptide (SEQ ID NO: 28)positioned between the α and β chains. The combined nucleotide sequencewas codon-optimized for expression in human tissues to provide a vectorinsert. The vector insert was cloned into an MSGV1 expression vector.The TCR encoded by the vector comprised an α chain comprising an aminoacid sequence comprising SEQ ID NOs: 9 and 24 and a β chain comprisingan amino acid sequence comprising SEQ ID NO: 27 (“Cys-modified TCR” or“Cys TCR”).

The TCR of Example 3 (LVL-modified TCR) was further modified to includea Cys substitution in the constant region of each of the α and β chainsas follows. The nucleotide sequence encoding the constant region of theα chain of the LVL-modified TCR of Example 3 (amino acid SEQ ID NO: 21)was modified to substitute the native Thr at position 48 with Cys. Thenucleotide sequence encoding the constant region of the β chain of theLVL-modified TCR of Example 3 (SEQ ID NO: 19) was modified to substitutethe native Ser at position 56 with Cys. The nucleotide sequencesencoding the α and β chains were cloned into a single nucleotidesequence with the nucleotide sequence encoding the R chain positioned 5′of the nucleotide sequence encoding the α chain and a nucleotidesequence encoding a picomavirus 2A peptide (SEQ ID NO: 28) positionedbetween the α and β chains. The combined nucleotide sequence wascodon-optimized for expression in human tissues to provide a vectorinsert (SEQ ID NO: 40). The vector insert was cloned into an MSGV1expression vector resulting in SEQ ID NO: 38. The TCR encoded by thevector comprised an α chain comprising an amino acid sequence comprisingSEQ ID NO: 26 and a R chain comprising an amino acid sequence comprisingSEQ ID NO: 27 (“LVL-Cys-modified TCR” or “LVL-Cys TCR”).

Example 5

This example demonstrates that modification of the recombinantexpression vector encoding the chimeric anti-HPV 16 E7 TCR of Example 2improved HPV 16 E7₁₁₋₁₉ tetramer binding and improved recognition of HPV16⁺ tumor cell lines.

PBL from two donors were transduced with one of the recombinantexpression vectors set forth in Table 1.

TABLE 1 amino acid SEQ ID NOs Position of α and β chain in TCR of TCRvector chimeric anti-HPV 16 E7 9, 10, 17, and 19, wherein alpha chain ispositioned 5′ of TCR (Example 2) X at position 2 of SEQ ID beta chain(“α/β”) NO: 10 is Ala Cys-modified TCR 9, 24, 27 α/β LVL-modified TCR20, 22 α/β LVL-Cys-modified TCR 26, 27 α/β chimeric anti-HPV 16 E7 9,10, 17, and 19, wherein beta chain is positioned 5′ of TCR (Example 2) Xat position 2 of SEQ ID alpha chain (“β/α”) NO: 10 is Ala Cys-modifiedTCR 9, 24, 27 β/α LVL-modified TCR 20, 22 β/α LVL-Cys-modified TCR 26,27 β/α

Transduced PBL from two normal donors were tested for HPV 16 E7₁₁₋₁₉tetramer binding by flow cytometry using anti-HPV 16 E7₁₁₋₁₉ tetramerand anti-mouse(m)TRBC antibodies on day 8 after stimulation.Untransduced cells were used as a negative control. The results areshown in FIGS. 2A-2R. The percentage of stained cells detected in eachquadrant is given above each graph. As shown in FIGS. 2A-2R, cellstransduced with a recombinant expression vector in which the β chain waspositioned 5′ of the α chain demonstrated improved tetramer binding ascompared to cells transduced with a recombinant expression vector inwhich the α chain was positioned 5′ of the R chain. As also shown inFIGS. 2A-2R, cells transduced with a Cys-modified TCR, a LVL-modifiedTCR, or a LVL-Cys-modified TCR each demonstrated improved tetramerbinding as compared to cells transduced with the TCR of Example 2.

In a separate experiment, transduced cells were co-cultured with 624cells, CaSki cells, Scc90 cells, or Scc152 cells 11 days afterstimulation, and IFN-γ secretion was measured. Untransduced cells wereused as a negative control. The results are shown in FIGS. 3A and 3B. Asshown in FIGS. 3A and 3B, cells transduced with a Cys-modified TCR, aLVL-modified TCR, or a LVL-Cys-modified TCR each demonstrated improvedrecognition of HPV 16⁺ tumor lines as compared to cells transduced withthe TCR of Example 2. As also shown in FIGS. 3A-3B, cells transducedwith a recombinant expression vector in which the β chain was positioned5′ of the α chain generally demonstrated improved recognition of HPV 16⁺tumor lines as compared to cells transduced with a recombinantexpression vector in which the α chain was positioned 5′ of the R chain.

Cells transduced with a recombinant expression vector in which the βchain was positioned 5′ of the α chain also demonstrated improvedexpression as compared to cells transduced with a recombinant expressionvector in which the α chain was positioned 5′ of the R chain, asmeasured by flow cytometry.

Example 6

This example demonstrates that T cells transduced with a recombinantexpression vector encoding the LVL-Cys-modified TCR (β/α) demonstratedCD8-independent binding of HPV-16 E7₁₁₋₁₉ tetramer.

Peripheral blood cells were transduced with the MSGV1 recombinantexpression vector encoding the LVL-Cys-modified TCR (β/α) of Example 4.The transduced cells were labeled with anti-CD8, anti-TRBC (mouseconstant region) antibodies, and HLA-A2/E7₁₁₋₁₉ tetramer and analyzed byflow cytometry. The CD8⁺ transduced cells and the CD8-transduced cellsfrom both donors both bound the tetramer, which demonstratedCD8-independent binding.

Example 7

This example demonstrates that peripheral blood T cells transduced toexpress the LVL-modified TCR (β/α) specifically recognize HPV 16 E7₁₁₋₁₉peptide and HPV 16⁺ tumor cell lines.

PBL were transduced with a recombinant expression vector encoding theLVL-modified TCR (β/α) (SEQ ID NO: 37) of Example 3. Rapid expansion ofthe numbers of cells was performed using the Rapid Expansion Protocol(REP) as previously described (Dudley et al. J Immunother. 26:332-42(2003) and Riddell et al. J Immunol. Methods 128:189-201 (1990)).Briefly, TIL were cultured with irradiated (40 Gy) allogeneic peripheralblood mononuclear “feeder” cells in complete medium (CM) with 30 ng/mLanti-CD3 antibody and 6000 IU/mL IL-2. The expanded, transduced cellswere co-cultured with target 293-A2 cells pulsed with HPV 16 E6₂₉₋₃₈peptide (control), 293-A2 cells pulsed with HPV 16 E7₁₁₋₁₉ peptide, 624cells transduced with a plasmid encoding HPV 16 E6, 624 cells transducedwith a plasmid encoding HPV 16 E7, SCC152 cells, SCC90 cells, CaSkicells, Alb cells, Panc1 cells, Ane cells or SiHa cells. IFN-γ wasmeasured. The results are shown in FIG. 4. As shown in FIG. 4, PBLtransduced with a recombinant expression vector encoding theLVL-modified TCR (β/α) specifically recognized HPV 16 E7-positive tumorcell lines and other HLA-A2⁺HPV16 E7⁺ targets in an HLA-A2-restrictedmanner. Results obtained with the cells of a second donor were similar.

Example 8

This example demonstrates that peripheral blood T cells transduced toexpress the LVL-Cys-modified TCR (β/α) specifically recognize HPV 16⁺tumor cell lines and that this recognition is blocked by anti-MHC ClassI antibodies. This example also demonstrates these transduced T cellsspecifically kill HPV 16⁺ HLA-A2⁺ tumor cell lines.

PBL were transduced with a recombinant expression vector encoding theLVL-Cys-modified TCR (β/α) (SEQ ID NO: 38). Rapid expansion of thenumbers of cells was performed using REP as described in Example 7.Transduced cells were co-cultured with target 624 cells, SCC90 cells, orCaSki cells without antibodies (black bars) or in the presence ofanti-MHC Class I (grey bars) or anti-MHC Class II antibodies (unshadedbars). As controls, PBL were transduced with DMF5 TCR and co-culturedwith 624 cells or transduced with anti-MAGE A3 TCR and co-cultured with526-CIITA cells without antibodies or in the presence of anti-MHC ClassI or anti-MHC Class II antibodies. IFN-γ was measured. The results areshown in FIG. 5. As shown in FIG. 5, peripheral blood T cells transducedto express the LVL-Cys-modified TCR (β/α) specifically recognized HPV16⁺ tumor cell lines and this recognition was blocked by anti-MHC ClassI antibodies.

In a separate experiment, transduced effector cells were co-culturedwith target CaSki cells, SCC90 cells, SCC152 cells, or 624 cells atvarious effector:target ratios. Untransduced effector cells were used asa negative control. The results are shown in FIGS. 6A-6D. As shown inFIGS. 6A-6D, PBL transduced with a recombinant expression vectorencoding the LVL-Cys-modified TCR (β/α) (SEQ ID NO: 38) demonstratedspecific killing of HPV 16⁺ HLA-A2⁺ tumor lines.

Example 9

This example demonstrates that peripheral blood T cells transduced toexpress the LVL-Cys-modified TCR (β/α) have functional avidity similarto the anti-HPV 16 E6 TCR DCA2E6. This example also demonstrates thatperipheral blood T cells transduced to express the LVL-Cys-modified TCR(β/α) demonstrated greater IFN-γ production upon co-culture with CaSkiand SCC152 cells but not SCC90 cells as compared to cells transducedwith the DCA2E6 TCR.

PBL were transduced with a recombinant expression vector encoding theLVL-Cys-modified TCR (β/α) (SEQ ID NO: 38) or DCA2E6. Transduced cellswere co-cultured with T2 cells pulsed with no peptide or concentrationsof HPV 16 E7₁₁₋₁₉ peptide or HPV 16 E6₂₉₋₃₈ peptide ranging from 1 μM to1 pM. IFN-γ was measured. The results are shown in FIGS. 7A and 7B. Asshown in FIGS. 7A and 7B, PBL transduced to express the LVL-Cys-modifiedTCR (0/a) have functional avidity similar to the anti-HPV 16 E6 TCRDCA2E6. Results obtained with the cells from a second donor weresimilar.

In a separate experiment, PBL were transduced to express theLVL-Cys-modified TCR (β/α) (SEQ ID NO: 38) or DCA2E6. Untransduced cellswere used as a negative control. Cells were co-cultured with target 624cells, Caski cells, SCC90 cells, or SCC152 cells. IFN-γ was measured.The results are shown in FIG. 8A. As shown in FIG. 8A, peripheral bloodT cells transduced to express the LVL-Cys-modified TCR (β/α)demonstrated greater IFN-γ production upon co-culture with CaSki andSCC152 cells but not SCC90 cells as compared to cells transduced withthe DCA2E6 TCR.

In a separate experiment, PBL were transduced to express theCys-modified TCR (β/α) (SEQ ID NOs: 9, 24, and 27) or DCA2E6.Untransduced cells were used as a negative control. Cells wereco-cultured with target 293-A2 cells pulsed with HPV 16 E6₂₉₋₃₈ peptide(control), 293-A2 cells pulsed with HPV 16 E7₁₁₋₁₉ peptide, SCC152cells, SCC90 cells, CaSki cells, Alb cells, Ane cells, Panc1 cells, orSiHa cells. IFN-γ was measured. The results are shown in FIG. 8B. Asshown in FIG. 8B, peripheral blood T cells transduced to express theCys-modified TCR (β/α) demonstrated greater IFN-γ production uponco-culture with CaSki and SCC152 cells but not SCC90 cells as comparedto cells transduced with the DCA2E6 TCR.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of producing an engineered population of human cells, the method comprising: introducing a recombinant expression vector to an isolated population of human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a T cell receptor (TCR) comprising the amino acid sequences of SEQ ID NOs: 3-8 and having antigenic specificity for human papillomavirus (HPV) 16 E7.
 2. The method of claim 1, wherein the TCR has antigenic specificity for HPV 16 E7₁₁₋₁₉ SEQ ID NO:
 2. 3. The method of claim 1, wherein the TCR comprises the amino acid sequences of: (a) SEQ ID NO: 9 and (b) SEQ ID NO: 10, wherein X at position 2 is Ala or Gly.
 4. The method of claim 1, wherein the TCR further comprises the amino acid sequences of: (a) SEQ ID NO: 16, wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 18, wherein X at position 56 is Ser or Cys.
 5. The method of claim 1, wherein the TCR further comprises the amino acid sequences of: (a) any one of SEQ ID NOs: 14, 17, 21, 24, and 25; and (b) any one of SEQ ID NOs: 15, 19, and
 23. 6. The method of claim 1, wherein the TCR comprises the amino acid sequences of: (a) (i) SEQ ID NO: 12, (ii) SEQ ID NO: 22, (iii) SEQ ID NO: 26, (iv) SEQ ID NO: 9 and 24, (v) SEQ ID NO: 9 and 16, or (vi) SEQ ID NOs: 9 and 17; and (b) (i) SEQ ID NOs: 10 and 18 or (ii) any one of SEQ ID NOs: 13, 20, and
 27. 7. The method of claim 1, wherein the TCR comprises a human variable region and a murine constant region.
 8. A method of producing an engineered population of human cells, the method comprising: introducing a recombinant expression vector to an isolated population of human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a polypeptide comprising a functional portion of a TCR, and wherein the functional portion comprises the amino acid sequences of (a) SEQ ID NOs: 3-5, (b) SEQ ID NOs: 6-8, or (c) SEQ ID NOs: 3-8.
 9. The method of claim 8, wherein the functional portion comprises the amino acid sequence of: (a) SEQ ID NO: 9; (b) SEQ ID NO: 10; (c) SEQ ID NOs: 9 and 10; (d) (i) SEQ ID NOs: 9 and 24; (ii) SEQ ID NOs: 9 and 17; (iii) SEQ ID NO: 9 and 16; (iv) SEQ ID NO: 10 and 18; or (v) any one of SEQ ID NOs: 12, 13, 20, 22, 26, 27, 29 and 30; (e) SEQ ID NOs: 12 and 13; (f) SEQ ID NOs: 20 and 22; (g) SEQ ID NOs: 26 and 27; (h) SEQ ID NOs: 9, 24, and 27; (i) SEQ ID NOs: 9, 17, and 20; or (j) SEQ ID NOs: 9, 10, 16, and
 18. 10. The method of claim 8, wherein the functional portion further comprises the amino acid sequences of: (a) SEQ ID NO: 16, wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 18, wherein X at position 56 is Ser or Cys.
 11. The method of claim 8, wherein the functional portion further comprises the amino acid sequences of: (a) any one of SEQ ID NOs: 14, 17, 21, 24, and 25; and (b) any one of SEQ ID NOs: 15, 19, and
 23. 12. A method of producing an engineered population of human cells, the method comprising: introducing a recombinant expression vector to an isolated population of human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a protein comprising a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 3-5 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 6-8.
 13. The method of claim 12, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10, wherein X at position 2 of SEQ ID NO: 10 is Ala or Gly.
 14. The method of claim 12, wherein: the first polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 16, wherein: (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and the second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 18, wherein X at position 56 is Ser or Cys.
 15. The method of claim 12, wherein: the first polypeptide chain further comprises the amino acid sequence of any one of SEQ ID NOs: 14, 17, 21, 24, and 25; and the first polypeptide chain further comprises the amino acid sequence of any one of any one of SEQ ID NOs: 15, 19, and
 23. 16. The method of claim 12, wherein: the first polypeptide chain comprises the amino acid sequence of (i) SEQ ID NO: 12, (ii) SEQ ID NO: 22, (iii) SEQ ID NO: 26, (iv) SEQ ID NO: 9 and 16, (v) SEQ ID NO: 9 and 17, or (vi) SEQ ID NO: 9 and 24 and the second polypeptide chain comprises the amino acid sequence of (i) SEQ ID NO: 10 and 18 or (ii) any one of SEQ ID NOs: 13, 20, and
 27. 17. The method of claim 1, wherein the isolated population of human cells is an isolated population of human peripheral blood lymphocytes.
 18. The method of claim 1, wherein the isolated population of human cells is an isolated population of human peripheral blood mononuclear cells.
 19. The method of claim 1, wherein the isolated population of human cells is an isolated population of human T cells.
 20. The method of claim 1, wherein the isolated population of human cells is an isolated population of human CD8⁺ T cells. 